Prevalence and assessment of risk factors for haemoprotozoan infections in cattle and buffaloes of South-West Gujarat, India

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1 Indian J. Anim. Res., 50 (5) 2016 : Print ISSN: / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Prevalence and assessment of risk factors for haemoprotozoan infections in cattle and buffaloes of South-West Gujarat, India B.R. Maharana*, Binod Kumar, A. Prasad 1, T.K. Patbandha 2, N.R. Sudhakar 3, J.P. Joseph 1 and B.R. Patel 1 Department of Veterinary Parasitology, College of Veterinary Science & A.H, JAU, Junagadh , India. Recieved: Accepted: DOI: /ijar ABSTRACT A total of 480 suspected blood samples were collected from cattle (n=254) and buffaloes (n=226) during the study period from September 2011 to December 2014 for determination of prevalence of haemoprotozoan parasites from South Western Gujarat (Junagadh). The conventional optical microscopy of Giemsa stained blood smears revealed that 37% of cattle and 38.93% of buffaloes were infected with haemoprotozoan parasites including, Babesia bigemina, Trypanosoma and. The animals infected with haemoprotozoan diseases showed lower total erythrocyte count (TEC), haemoglobin (Hb), packed cell volume (PCV), mean corpuscular haemoglobin concentration (MCHC) and higher total leukocyte count (TLC) and mean corpuscular volume (MCV). Incidence of haemoprotozoan infection between the breeds differed significantly (P<0.05). Haemoprotozoan infections were recorded significantly higher (P<0.001) in monsoon season followed by summer and being lowest in winter season in cattle. Over all prevalence in female animals were higher than male counterparts. In both the species, over all higher prevalence of babesiosis was recorded in comparison to other haemoprotozoan diseases and the difference being statistically non-significant (p>0.05). The prevalence of B. bigemina in bovines was associated with various risk factors namely age, season and breed (p 0.05). Sex wise females recorded apparently higher infection rates than males. The multivariate logistic regression models showed that the risk of babesiosis was significantly higher in rainy season (OR=5.18, P=0.003) followed by summer (OR=3.9, P=0.019) compared to winter season. Conversely, in buffaloes, the risk of babesiosis was significantly higher in summer season (OR=9.0, P=0.004) followed by rainy (OR=7.43, P=0.008) compared to winter season. The risk for anaplasmosis in buffaloes increased by 3.46 times (OR=3.64, p=0.027) in non descriptive breed compared to well defined breed. Key words: es,, Haemoprotozoan parasites, Prevalence, Risk factor. INTRODUCTION Haemoprotozoan parasites cause economically important vector-borne diseases of tropical and subtropical parts of the world including India (Salih et al., 2015). The hot and humid climate is favorable for the development and survival of potential vectors which in turn act as a constant source of infection to susceptible animals (Velusamy et al., 2014). They cause significant morbidity and mortality in cattle and buffaloes. Dairy animals, especially bovines, which are bearing production stress along with other diseases, are potential viable host to these infections (McLeod and Kristjanson, 1999; Sharma et al., 2013). An estimate of US$ million per annum was calculated as the cost of controlling ticks and tick borne diseases in India (Minjauw and McLeod, 2003). Further, in India, tropical theileriosis has alone claimed an annual loss of approximately US $ 800 million (ICAR, 2005). Prevalence of Haemoprotozoan parasites has been reported in animals of different parts of India (Agrawal et al., 2003; Muraleedharan et al., 2005; Shahnawaz et al., 2011; Vahora et al., 2012; Arun Kumar et al., 2013; Velusamy et al., 2014; Kohli et al., 2014; Sharma et al., 2015a). However, true status of haemoprotozoan infections has not yet been explored in bovines in and around Junagadh, South western Gujrat of India which is a rich source of livestock with breeds like Gir cattle, Jaffrabadi buffalo and the agroecological and geo-climatic conditions of this region are highly favourable for growth and multiplication of natural vectors of these diseases. So, it is the need of the hour, to have a thorough study regarding the seasonal occurrence of parasitic infections in order to go for a targeted selective treatment and measures to prevent the outbreaks without giving much economic and financial burden on the part of the farmers. Therefore, the present study was designed to investigate the seasonal prevalence of haemoprotozoan parasites in bovines by conventional gold standard diagnostic method with the aim to obtain baseline data on various risk factors to design effective control strategy. *Corresponding author s drbiswaranjanmaharana@gmail.com 1 Department of Veterinary Medicine, 2 Polytechnic in Animal Husbandry, 3 Department of Veterinary Parasitology. College of Veterinary Science & A.H., SVPUA &T, Meerut (U.P.), India.

2 734 INDIAN JOURNAL OF ANIMAL RESEARCH MATERIALS AND METHODS Geographical area: The study was conducted in and around Junagadh district of Gujarat state, in the south-western region of India. Junagadh has a tropical wet and dry climate, with three distinct seasons observed, a mild winter from November to February, a hot summer from March to June, and a monsoon from July to October. Junagadh faces adverse climatic conditions in the summer months with the temperature ranges from 28 C to 38 C. In the winter months, the temperature ranges from 10 C to 25 C. The city is drained by the south-west monsoon from June to September. It records a rainfall of 1000 to 1200 mm annually. These environmental conditions provide favourable and conducive conditions for the survival and propagation of vectors which play an important role in transmission of haemoprotozoan diseases. Study Period: The data recorded for a period of 3 years and 4 months from September 2011 to December Sample collection: A total of 480 blood samples of bovines were collected at Teaching Veterinary Clinical Complex (TVCC), Veterinary College Junagadh, Breeding Farm (CBF), Junagadh, Veterinary Poly Clinic, Junagadh and different animal camps organized by Veterinary College in villages in and around the Junagadh district. The blood samples were collected from ear vein and Jugular vein of the animals which were suspected for haemoprotozoan infection based on clinical signs of fever, anaemia, lymph node swelling etc. For blood smear preparation, ear vein was punctured by 18G needle and drop of blood was smeared on the grease free micro slide, dried and wrapped in a clean paper. Whole blood was collected from Jugular vein with the help of sterile 5 ml syringe and reserved in a vial containing EDTA and kept on ice. This whole blood was also used for smear preparation and other hematological examination. Animal data was recorded in a proforma containing information like owner name, place, species, breed, age, date etc. Processing of Blood Samples: The Giemsa stained smears were examined under oil immersion (100X) for any haemoprotozoan parasites. For study of various hematological parameters, viz. total erythrocyte count (TEC), haemoglobin (Hb), total leucocyte count (TLC), differential leucocyte count (DLC), packed cell volume (PCV), thrombocyte count and erythrocyte indices such as mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), blood auto-analyzer was used. Statistical analysis: Data obtained were compiled and tabulated for frequency and finally converted into percentage to draw inferences. Chi-square ( 2 ) test was used to see significant differences (P 0.05) in parasitic infections among age, sex, breed and season. Parameters which had significant effect on incidence of haemoprotozoan were further subjected to multivariate logistic regression models. The logistic regression models produce Odds ratios (OR) as estimates of the strength of association between the potential risk factors (age, breed and season) and incidence of haemoprotozoan parasites. Statistical analysis was carried out using SPSS software package (Version 17.0, USA). RESULTS AND DISCUSSION Incidence of haemoprotozoan parasite infections: Out of 480 blood samples (254 from cattle and 226 from buffaloes) examined, Giemsa stained blood smears revealed an overall haemoprotozoan prevalence of 37 and 38.93% in cattle and buffaloes, respectively. Babesiosis was highest (22.83%), followed by theileriosis (7.08%), anaplasmosis (3.93%) and trypanosomosis (3.14%) in cattle (Table 1). In buffaloes, babesiosis, anaplasmosis, trypanosomosis and theileriosis was 20.35, 7.07, 6.19 and 5.30%, respectively (Table 1). Our observations correlate with the results of previous studies on incidence of haemoprotozoan parasites in bovines. (Roy et al., 2004; Ananda et al., 2009, 2014; Sharma et al., 2015 a & b). In the present study, highest incidence of B. bigemina infection was recorded in both cattle and buffaloes. The prevalence depends largely on the distribution and density of the reservoir hosts season and vectors (Singh et al., 2000; Ogden et al., 2002).The higher prevalence of bovine babesiosis can be closely correlated to the seasonal abundance of the vector Rhipicephalus microplus (Singh et al., 2000). The lower prevalence of theileriosis in bovines may be due to the lower occurrence of vector Hyalomma anatolicum anatolicum in this geographical part of the country. Though, blood samples were collected from suspected animals, the rate of infections was very low. This might be because of various factors which reduce the chance of parasite detection in blood smear like very low sensitivity of microscope, time of blood collection, site of blood collection etc. (Tuli et. al., 2015). Analysis of hematological parameters: The animals infected with haemoprotozoan disease had lower TEC, Hb, PCV, MCHC and TLC, MCV, lymphocyte and higher neutrophil count (Table 2). This might be due to damage caused by the heamoprotozoan organisms inside the RBC s during their multiplication (Soulsby, 1982). The present report of haematological alternation in haemoprotozoan infection is in conformity with the observation made by Table 1: Incidence of haemoprotozoan parasites in cattle (n=254) and buffalo (n=226) of Junagadh. Haemoprotozoan parasite No. of positive samples (% of incidence) 10 (3.93) 16 (7.07) Babesia bigemina 58 (22.83) 46 (20.35) 18 (7.08) 12 (5.30) Trypanosoma 8 (3.14) 14 (6.19) Total 94 (37.00) 88 (38.93)

3 Table 2. Comparison of hematological parameters of normal, suspected and infected with haemoprotozoan disease in cattle and buffalo. (Mean ±SEM) Volume 50 Issue 5 (2016) 735 Note: TEC - Total Erythrocyte count, Hb - Haemoglobin, TLC - Total leucocyte count, DLC - Differential leucocyte count, PCV - Packed cell volume, TC - Thrombocyte count, MCV - Mean corpuscular volume, MCHC - Mean corpuscular haemoglobin concentration, L- Lymphocyte, E-Eosinophil, M-Monocyte, B-Basophil, N-Neutrophil. earlier workers (Muraleedharan et al., 2005; Singla et al., 2013; Singh et al., 2014).The data for the hematological parameters for suspected animals are coming within the range of normal and infected animals with high standard error (SE). This shows the very high animal to animal variation. These data indicating that the suspected animals might be a carrier of disease or having very milder infection which is not detectable under microscope. Incidence of haemoprotozoan parasite infections in relation to risk factors: Age wise animals were divided in to three groups viz. young (below 1 year), adults (1 to 5 yrs) and old (more than 5 yrs). Data was also recorded sex wise (male and female), breed wise (Gir cattle, Jaffrabadi buffalo and non-descriptive breed), and season wise (summer: March to June, monsoon: July to October and winter: November to February). There was significant difference in overall prevalence of haemoprotozoan infection in relation to various age groups in both cattle and buffaloes (p<0.05) (Table 3). Age wise prevalence of anaplasmosis and theileriosis were higher in older group followed by adult group of cattle. Babesiosis and trypanosomosis were recorded highest in adult group of cattle in comparison to older ones. The present study revealed significant statistical difference between the different age group of cattle in relation to the prevalence of babesiosis and theileriosis. In case of buffaloes, higher incidence of haemoprotozoan parasites was recorded in adult age group with the exception of spp. being higher in older age group. Further, the study revealed significant difference (p<0.05) in incidence of babesiosis in different age group of buffaloes. Inverse age resistance and stronger passively acquired immunity might be the contributing factors behind the lower incidence of haemoprotozoan parasites in younger age groups of animals. Breed wise higher infection rates were documented in non-descript breeds than pure breeds in both cattle (42.46% vs 29.62%) and buffaloes (51.72% vs 29.09%) (Table 4). Among all the recorded haemoprotozoan infections, B. bigemina was in higher proportion irrespective of the breeds of bovines (Table 4). In case of buffaloes, significantly higher prevalence of A. was recorded in non-descript breed than pure breeds (12.07% vs 3.64%, p<0.05). Difference in innate and acquired immunity, animal husbandry and managemental practices might be the contributing factor towards higher prevalence of haemoprotozoan infections in ND breeds compared to pure breed (Singla et al., 2013; Velusamy et al., 2014; Bhatnagar et al., 2015; Sharma et al., 2015 a &b). Season wise overall prevalence was recorded highest in monsoon followed by summer and winter in cattle (Table 5), the data being statistically significant (p<0.05). But in case of buffaloes, the overall prevalence was

4 736 INDIAN JOURNAL OF ANIMAL RESEARCH Table 3: Incidence (%) of haemoprotozoan parasitic infections according to age of the animals. Age Group (Total sample) Babesia bigemina Trypanosoma prevalence Young (40) 0(0) 0 (0) 0 (0) 5 (2) 5 (2) Adult (134) 4.47 (6) (38) 5.97 (8) 2.98 (4) (56) Old (80) 5 (4) 25 (20) 12.5 (10) 2.5 (2) 45 (36) χ 2 value *** 6.86** *** Young (36) 0 (0) 0 (0) 0 (0) 5.55 (2) 5.5 (2) Adult (122) 9.83 (12) (30) 4.92 (6) 8.19 (10) (58) Old (68) 5.88 (4) (16) 8.82 (6) 5.88 (4) (30) χ 2 value ** *** Table 4: Incidence of haemoprotozoan parasitic infections according to breed of the animals. Animal Breeds (Total sample) Babesia bigemina Trypanopsoma prevalence Gir 1.85 (2) (32) 3.70 (4) (20) 5.56 (6) ( 108) ND 4.11 (6) (62) 4.11 (6) (38) 8.22 (12) (146) χ 2 value * Juffarabadi 5.45 (6) (32) 3.64 (4) (18) 3.64 (4) (110) ND 8.62 (10) (60) (14) (28) 6.89 (8) (116) χ 2 value 4.38* *** Table 5: Incidence of haemoprotozoan parasitic infections according to season of the animals. Season (Total samples) Babesia bigemina Trypanosoma prevalence Summer (84) 4.76 (4) (20) 7.14 (6) 2.38 (2) (32) Monsoon (116) 5.17 (6) (34) 8.62 (10) 3.45 (4) (54) Winter (54) 0 (0) 7.41 (4) 3.70 (2) 3.70 (2) (8) χ 2 value ** *** Summer (66) 9.09 (6) (18) 6.06 (4) 9.09 (6) (34) Monsoon (110) 9.09 (10) (26) 7.27 (8) 5.45 (6) (50) Winter (50) 4.0 (2) 4.0 (2) 0 (0) 4.0 (2) 12.0 (6) χ 2 value ** *** significantly higher in summer season followed by monsoon and lowest in winter season. Among all haemoprotozoans, the occurrence of B. bigemina (both cattle and buffaloes) was highest in monsoon season followed by summer and winter season, the data being differed statistically (p<0.01). However, there was no significant seasonal influence on the comparative distribution of anaplasmosis, theileriosis and trypanosomosis in bovines (p>0.05). Most of the animals suffering during summer and monsoon months might be due to high abundance of vectors in these seasons of the year (Bhatnagar et al., 2015; Velusamy et al., 2015; Radostits et al., 1994; Roy et al. 2004).

5 incidence of haemoprotozoan parasite infection in female cattle (40.44%) was higher than male (28.94%) counterparts and a similar trend was observed for buffaloes (40.74% vs 28.12%), but did not differ significantly (p>0.05) owing to common managemental practices being adopted for both the sexes (Table 6). But higher prevalence in female population may be due to hormonal disturbances which pretense it to weakened immune system. Correlation of occurrence of haemoprotozoan infection with various risk factors: The multivariate logistic regression models (Table 7) showed that risk of babesiosis was significantly higher in rainy season (OR=5.18, P=0.003) followed by summer (OR=3.9, P=0.019) compared to winter season in cattle. Conversely, in buffaloes, risk of babesiosis was significantly higher in summer season (OR=9.0, P=0.004) followed by rainy (OR=7.43, P=0.008) compared to winter season. The risk for anaplasmosis in ND buffaloes increased by 3.46 times (OR= 3.64, P=0.027) compared to well defined breeds. Compared to young cattle, the risk of overall prevalence of haemoprotozoan infection was significantly higher in older (OR=1.140, p=0.646) and lower Table 6: Incidence of Haemoprotozoan parasitic infections according to sex of the animals. Sex (Total samples) Volume 50 Issue 5 (2016) 737 Babesia bigemina Trypanosoma Prevalence Male (2) (16) 5.26 (4) (76) (0) (22) Female cattle 4.49 (8) (72) 4.49 (8) (42) 7.86 (14) (178) χ 2 value Male 3.12 (2) (18) 6.25 (4) (10) 3.12 (2) (64) Female 4.94 (8) (66) 7.41 (12) (36) 6.17 (10) (162) χ 2 value Table 7: Risk factors associated with haemoprotozoan infection in bovines Parameters Estimates SEM P value OR 95% CI Babesia: Age (old vs adult) Season (summer vs winter) Season (rainy vs winter) Babesia: Age (old vs adult) Season (summer vs winter) Season (rainy vs winter) : Age (old vs adult) : Breed ND vs Descriptive SEM, Standard error mean; OR, Odds Ratio; CI, Confidence interval; ND, Non descriptive in adult (OR=0.073, p<0.001) (Table 8). Additionally, the risk of occurrence of overall haemoprotozoans was significantly higher in ND breeds of cattle (OR=1.753, p=0.037) compared to purebred. Rainy (OR=5.008, p<0.001) and summer (OR=3.538, p=0.004) season acted significant risk factor for overall prevalence of haemoprotozoan infection compared to winter season in cattle. In buffaloes, the risk of overall prevalence of haemoprotozoan infection was significantly higher in older ones (OR=0.871, p=0.650) but lower in adult (OR=0.065, p<0.001) than young ones. In ND buffalo breeds, the risk of occurrence of overall haemoprotozoans was significantly higher (OR=2.612, p=0.001) compared to pure breeds. The risk of prevalence of overall haemoprotozoan infection was observed to be significantly higher in summer season (OR=7.792, p<0.001) followed by rainy (OR=6.111, P<0.001) when compared to winter season (Table 8). The various risk factors discussed above significantly associated with the occurrence of disease. Our findings are not indifferent from those reported elsewhere (Sajid et al., 2014; Atif et al., 2012a; Atif et al., 2012b ).

6 738 INDIAN JOURNAL OF ANIMAL RESEARCH Table 8: Risk factors associated with overall haemoprotozoan infection in bovines Parameters Estimates SEM P value OR 95% CI : haemoprotozoan Age (Young vs adult) < Age (old vs adult) Breed ND vs Descriptive Season (summer vs winter) Season (rainy vs winter) < : haemoprotozoan Age (Young vs adult) < Age (old vs adult) Breed ND vs Descriptive Season (summer vs winter) < Season (rainy vs winter) < SEM, Standard error mean; OR, Odds Ratio; CI, Confidence interval; ND, Non descriptive CONCLUSION The upshot of the present investigation would be of enormous help for the dairy farmers, policy and decision makers in forecasting outbreak and formulating effective target selective chemoprophylactic approach not only in this region in specific but also to other parts of the country in general. Simultaneously, there is an urgent need for further effective diagnosis of concurrent latent haemoprotozoan infections using advanced molecular techniques. ACKNOWLEDGMENTS The authors are highly thankful to Director of Research and Dean P.G. studies, J.A.U., Junagadh for approval of the project in 18 th and 20 th ZEARC to carry out this pilot study on bovines. Authors are highly thankful to Principal and Dean, College of Veterinary Science & A.H., Junagadh for providing the necessary facilities. The facilities provided by TVCC, Veterinary College, Junagadh are thankfully acknowledged. REFERENCES Agrawal, R., Singh, R., Kumar, M. and Upadhyay, A.K. (2003). Epidemiological features of bovine trypanosomiosis and babesiosis in Durg district of Chhattisgarh state. Indian Vet. J. 80: Ananda, K.J., Ganganaik, S. and Kavitha, R.B. (2014). Epidemiological studies on haemoprotozoan diseases in bovines in and around Shimoga: a malnad region of Karnataka. Indian Vet. J. 91: Ananda, K.J., Placid, E, D Souza. and Puttalakshmamma, G.C. (2009). Prevalence of haemoprotozoan diseases in crossbred cattle in Bangalore north. Vet. World. 2: Arunkumar, S. and Nagarajan, K. (2013). A study on prevalence status of infection among cattle population of Kancheepuram and in and around Chennai districts of Tamil Nadu. Int. J. Food Agri. and Vet. Sci. 3: Atif, F. A., Khan, S., Iqbal, H.J. and Roheen, T. (2012a). Prevalence of tick-borne diseases in Punjab, Pakistan and hematological profile of infection in indigenous and crossbred cattle. Pakistan J. Sci. 64: Atif, F.A., Khan, M.S., Iqbal, H.J., Arshad, G.M., Ashraf, E. and Ullah, S. (2012b). Prevalence of, Babesia bigemina and infections among cattle in Sargodha District, Pakistan. Afr. J. Agri. Res. 7: Bhatnagar, C.S., Bhardawaj, B, Sharma, D.K. and Meena, S.K. (2015). Incidence of Haemoprotozoan diseases in cattle in Southern Rajasthan, India. Int. J. Curr. Microbiol. App. Sci. 4: Kohli, S., Atheya, U.K. and Thapliyal, A. (2014). Prevalence of theileriosis in cross-bred cattle: its detection through blood smear examination and polymerase chain reaction in Dehradun district, Uttarakhan d, India. Vet. World. 7: Mallick, K.P., Dwivedi, S.K., Srivastava., N.K. and Kumar, S. (1987). A report on the occurrence of haemoprotozoan infection in rural live-stock. Indian J. Parasitol. 11: McLeod, R. and Kristjanson, P. (1999). Final Report of Joint ESYS/International Livestock Research Institute/ Australian Centre for International Agricultural Research Tick Cost Project-Economic Impact of Ticks and Tick-Borne Diseases to Livestock in Africa, Asia and Australia International Livestock Research Institute, Nairobi, Kenya.

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